Safety evaluation and application of lactic acid bacteria and yeast strains isolated from Sichuan broad bean paste

Abstract Broad bean paste is one of the most popular characteristic traditional fermented bean products in China, which is prepared by mixed fermentation of a variety of microorganisms, among which lactic acid bacteria and yeast played an important role in the improvement of the fermented broad bean paste quality. However, the traditional open‐air fermentation of broad bean paste brought some risks of harmful microorganisms. In this study, the safety and fermentation ability of lactic acid bacteria and yeast strains isolated from traditional broad bean paste was evaluated. The results showed that the protease activity of the strain Lactobacillus plantarum DPUL‐J5 (366.73 ± 9.00 U/L) and Pichia kudriavzevii DPUY‐J5 (237.18 ± 10.93 U/L) were the highest. Both strains produced little biogenic amines, and did not exhibit α‐hemolytic activity or antibiotic resistance for some of the antibiotics most used in human medicine. Furthermore, the broad bean paste fermentation involving DPUL‐J5 and DPUY‐J5 was beneficial for accumulating higher total acid (1.69 ± 0.01 g/100 g), amino‐acid nitrogen (0.85 ± 0.03 g/100 g), and more volatile flavor compounds, meanwhile, reducing the levels of biogenic amines and aflatoxin B1. Therefore, this study provided a new strategy to improve the safety and quality of traditional broad bean paste.

Traditional broad bean paste was fermented in the open air; there may be some safety problems such as harmful microorganism in the fermentation process. At the same time, some microorganisms in the broad bean paste can produce harmful substances such as aflatoxins B1 (AFB1) and biogenic amines (BAs), which may pose a threat to human health. Therefore, it is very necessary to evaluate and screen safe starter cultures to improve the safety and quality of the fermentation product. AFB1 is well recognized as one of the most critical mycotoxins and human carcinogens, which was produced by a genus of Aspergillus and commonly observed in numerous crops and fermented food (Zhang et al., 2020). BAs are organic nitrogenous compounds produced by microorganisms in food through the decarboxylation of an amino acid (Ruiz-Capillas & Herrero, 2019;Saaid et al., 2009). The formation of biogenic amines has two conditions, one was free amino acids and another was amine-producing microorganisms (Linares et al., 2012), which were especially rich in fermented bean products.
Lactic acid bacteria were reported to be strong BA producers in different fermented foods, and yeast produced fewer amines (Spano et al., 2010). Some molds also can produce biogenic amines, and Aspergillus oryzae and Mucor isolated from traditional Sichuan bean paste can produce tyramine (Hao & Sun, 2020). Although Bacillus can be used as a starter culture, some species can also produce biogenic amines, and their safety needs to be evaluated (Jae-Hyung et al., 2019). In addition to producing BAs, the starters have potential risks of having antibiotic resistance and hemolysis. It is imperative to screen microorganisms with no antibiotic resistance before using them as starters (Imperial & Ibana, 2016). Moreover, strains used in food production were required not to show hemolytic activity (Marty et al., 2012).
In this study, the safety and fermentation ability of lactic acid bacteria and yeast isolated from traditional broad bean paste were evaluated, and they were applied to the fermentation of broad bean paste, which provides a new strategy for improving the quality of broad bean paste and reducing risks.

| Strains and cultural conditions
In this study, LAB and yeast strains were isolated from traditional broad bean paste from the Pidu district (Chengdu city, Sichuan Province, China). There were 16 strains of LAB, which were cultured on Man, Rogosa, and Sharpe (MRS) medium for 18 h, and there were 14 yeasts which were cultured on yeast extract peptone dextrose (YPD) medium for 18 h. Aspergillus flavus DPUM-J1 and Bacillus subtilis DPUL-J2 were also isolated from traditional broad bean paste and did not produce AFB1 or BAs. Aspergillus and Bacillus had high protease and amylase activities and have been used as starters in fermented soybean paste . All strains were preserved in the Dalian probiotics function research key laboratory, Dalian Polytechnic University laboratory.

| The determination of the biogenic aminesproducing ability of the lactic acid bacteria and yeast strains
The production of BAs by LAB and yeast strains was determined according to the reported methods . The LAB and yeast strains were inoculated in the modified MRS or YPD broth which contained seven 0.1% (w/v) BA precursors, including L-tyrosine, L-histidine hydrochloride, L-lysine hydrochloride, L-tryptophan hydrochloride, L-arginine hydrochloride, L-ornithine hydrochloride, L-phenylalanine hydrochloride (Beijing Solarbio Science & Technology), and 0.005% (w/v) pyridoxal-5-phosphate-HCl (Sigma-Aldrich). The LAB were cultured at 37°C and the yeast at 28°C for 48 h in the modified MRS or YPD broth, respectively.
The 750 μl of each culture suspension was mixed with 750 μl dansyl chloride (Sigma-Aldrich) and 150 μl saturated sodium bicarbonate. The mixture was incubated for 30 min at 40°C. Residual dansyl chloride was removed by adding 150 μl ammonium hydroxide and incubated at 40°C for 15 min, complemented to 1.5 ml with acetonitrile through a 0.22 μm syringe filter, and analyzed by HPLC equipped with a Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) at 254 nm. The injection volume for the HPLC system was 30 μl. The flow rate was 1.0 ml/min, and the temperature was set at 40°C.

| The determination of protease activities of lactic acid bacteria and yeast strains
After previous laboratory studies, amylase and lipase activities of lactic acid bacteria and yeast were low, so only protease activity was measured. Five grams of broad bean and 50 ml of 2.5 mol/L NaCl solution were added into a conical flask and sterilized at 121°C for 20 min to prepare the fermentation medium. LAB and yeast were 2% inoculated into the fermentation medium and cultured at 37°C and 28°C for 48 h, respectively. After cultivation, the culture of LAB and yeast were centrifuged at 8000 × g (7630 rpm in a FA-45-6-30 rotor, centrifuge 5804R, Eppendorf AG) in 4°C for 10 min to obtain supernatant as a crude enzyme solution for determining protease activity by using Total Protease (Protease) ELISA Kit (BIO).

| Identification
The strain with high protease activity and extremely low BAs production was identified by 16S rDNA and 26S rRNA gene sequences analysis. The gene sequences were compared on NCBI using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for homology comparison, the recognized standard sequence data were obtained from the Gen Bank database, and the MAGE (v. 7.0, https://www.megas oftwa re.net/) was used to make a phylogenetic tree.

| Hemolytic activity test
Hemolysis activity was determined according to the method described by Jeong et al. (2014). The LAB and yeast were streaked on the Columbia agar medium (Oxoid Ltd) containing 5% (v/v) sheep blood (Beijing Solarbio Science & Technology Co., Ltd) and cultured at 37°C and 30°C for 48 h to detect patterns of hemolysis. The appearance of a grass-green ring indicated ɑ-hemolytic activity; the appearance of a well-defined and transparent hemolytic ring indicated β-hemolytic activity, and no change was no hemolysis. Listeria monocytogenes SHBCC D15669 (Shanghai Bioresource Collection Center) was used as a positive control for hemolytic analyses. Three independent experiments were conducted.

| Antibiotic susceptibility test
The antibiotic resistance profiles of the LAB and yeast were conducted by the method described by Nami et al. (2014)

| Broad bean paste preparation
The fermentation process of broad bean paste mainly included three stages: preparation of seed koji, preparation of broad bean koji, and mixed fermentation with brine (Zhu et al., 2017). Firstly, Aspergillus flavus DPUM-J1 was inoculated in potato dextrose agar (PDA) medium and cultured at 28°C for 72 h to obtain spores as seed koji.
Subsequently, the shelled broad beans were soaked, blanched, dried, mixed with the flour evenly at the ratio of 1:0.3 (w/w), and then the mixture was inoculated with Aspergillus flavus DPUM-J1 spore suspension (inoculation amount for 1.5%, v/w), cultivated at 30°C for 48 h as koji. The koji was turned once a day in order to prevent the koji from clumping until the surface of the broad bean was coated with A. flavus DPUM-J1 mycelium. Finally, the prepared broad bean koji was inoculated with 2% Bacillus subtilis DPUL-J2 and divided into four groups according to Figure 1, mixed with brine in a 1:1 ratio (the salt content of 16%, v/v), 2% inoculated with different strains for fermentation, covered the bottle with gauze, and fermented for 30 days at 30°C. During the fermentation process, stirred for every 5 days. Samples of the fermented broad bean pastes were taken on the 0, 5, 10, 15, 20, 25, and 30 days for chemical properties analysis.

| Chemical properties analysis of broad bean paste
The pH level of the broad bean paste samples was determined by pH meter. Additionally, the amino acid nitrogen (AAN) and total acidity (TA) contents of the broad bean paste were analyzed according to the National Standards. The reducing sugar content was determined by the 3,5-dinitrosalicylic acid (DNS) method (Wu et al., 2018). Aspergillus flavus DPUM-J1 was used to prepare koji. The material-to-liquid ratio was 1:1 (w/v) and submerged into a brine solution (16% w/v, NaCl). The suspension concentration of strain was 10 8 -10 9 CFU/ml, and the inoculation amount was 2% (v/v) 2.9 | The determination of biogenic amines and aflatoxin B1 content in broad bean paste Biogenic amines content in broad bean paste samples was detected in accordance with the methods described by Liu et al. (2021) and Kim and Ji (2015) with a slight modification. Five grams of broad bean paste sample was accurately weighed from different fermentation periods, and 20 ml of 5% trichloroacetic acid solution was added to remove protein; the mixture was homogenized using an orbital mixer for 60 min and centrifuged at 8000 × g (7630 rpm) at 4°C for 10 min to collected the supernatant, and the residues were mixed with 5% trichloroacetic acid and centrifuged again. The supernatant was merged twice and adjusted to 50 ml with 5% trichloroacetic acid, took 10 ml of the above extract and added an equal volume of n-hexane (Guangdong Guanghua Sci-Tech Co., Ltd) to remove lipid, vortexed for 5 min, and discarded the upper organic phase. 750 μl of the extract was transferred to a tube， added 150 μl of saturated sodium carbonate solution and 750 μl of dansyl chloride, and incubated at 45°C for 30 min to derive. Finally, 150 μl ammonia water was added and incubated at 45°C for 15 min. HPLC conditions and determination methods are described in 2.2.
Extraction of AFB1 from broad bean paste samples was performed according to the instruction of the producer supplied with the quantitative AFB1 test kits (CUSABIO, Wuhan, China).

| The determination of volatile flavor compounds
The volatile flavor compounds in broad bean paste samples fermented for 30 days were determined and extracted using the solid-phase microextraction (SPME) method according to Hu et al. (2021) and Kum et al. (2015). Three grams sample was taken and mixed with 10 μl cyclohexanone (Guangdong Guanghua Sci-Tech) solution (10 μg/ml), which was an internal standard in a 20 ml headspace vial at 60°C water bath for 30 min. Inserting the SPME extraction head (75 μm CAR/PDMS) with adsorbed volatile compounds into the headspace vial, the extraction head was directly injected into the injector of gas chromatography (GC)-mass spectrometry (GC-MS, Agilent Technologies Inc.) after 30 min. The sample was desorbed for 10 min at 250°C, and each sample was repeated three times. GC conditions: a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm) was used with helium as carrier gas at a flow rate of 1.0 ml/min. The heating procedure was as follows: the column temperature was 35°C initially, maintained for 3 min, then increased to 50°C at 3°C/min, followed by a 6°C/min rise to 150°C, raised to 230°C at 10°C/min, then held at

| Statistical analysis
All experiments were conducted in triplicate, and the statistical data were shown as the mean value ± standard deviation. SPSS 19.0 software package (SPSS Statistics) was used for one-way analysis of variance (ANOVA) and Duncan's test (α = .05).

| The LAB and yeast BA-producing test in vitro
The BA-producing ability of 16 LAB isolated from traditional Sichuan broad bean paste was determined to assess their safety. As shown in Table 1, putrescine, histamine, cadaverin, and β-phenylethylamine were detected in tested strains. Most of the strains produced putrescine and histamine, indicating that these strains had ornithine and histidine decarboxylase activity. The strain with strong amineproducing ability has the high activity of amino acid decarboxylase (Mah et al., 2019). Different kinds of microorganisms may produce different amino acid decarboxylases, which in turn, produce different biogenic amines. LAB were the main microorganisms that produced biogenic amines, among which the most common biogenic amines-producing bacteria genus were Enterococcus (Jeon et al., 2018), Lactobacillus (Li et al., 2016), and Lactococcus (Ladero et al., 2011). Biogenic amines formation in broad bean paste was most likely related to the activity of LAB.
Histamine and tyramine were the most dangerous BAs and are responsible for symptomatology known as "scombroid fish poisoning" and "cheese reaction," respectively (Hungerford, 2010;McCabe-Sellers et al., 2006). As mentioned by Yang et al. (2014), the maximum allowable concentration of histamine and tyramine range were 50-100 mg/kg and 100-800 mg/kg on food; beyond this range could cause migraines or severe poisoning symptoms. Strain DPUL-SC11 had the strongest ability to produce biogenic amines, and the total biogenic amine content was 7.97 ± 0.26 mg/kg (putrescine 3.39 ± 0.16 mg/kg and histamine 4.57 ± 0.23 mg/kg). Strain with strong biogenic amineproducing capacity was not suitable for starter culture in broad bean paste. Some strains had low biogenic amine-producing capacity such as DPUL-SC1, DPUL-SC3, DPUL-J5, and DPUL-SC9. The strain with extremely low or no BAs was selected as a potential starter culture to improve the safety of broad bean paste.
The highest BAs content in the four yeasts was DPUY-SC1 with a total BAs concentration of 9.02 ± 0.02 mg/kg and the content of β-

| Protease activity of LAB and yeast
During the fermentation process of broad bean paste, microorganisms will generate protease to catalyze the hydrolysis of polypeptides or proteins in the raw materials to generate various amino acids and increase the flavor components. The protease activity of the LAB and yeast strains in broad bean broth was measured as shown in Tables 3 and 4. The DPUL-J5 showed the highest protease activity, which was 366.73 ± 9.00 U/L, and the DPUY-J5 also showed the highest protease activity, which was 237.18 ± 10.93 U/L.

| Identification of the isolates
According to the BA-producing ability and protease activity results, LAB DPUL-J5 and yeast DPUY-J5 were selected for fermenting  The phylogenetic tree of them is shown in Figure 2.

| Antibiotic resistance
The antibiotic resistance properties of LAB strains about 10 common clinical antibiotics were evaluated according to the CLSI standards and the data were presented in Table 5. The results revealed that L. plantarum DPUL-J5 was sensitive to clindamycin (CLDM), erythromycin (ERY), ampicillin (AMP), and chloramphenicol (CAP) antibiotics, and resistant to vancomycin (VAN) antibiotics. Ammor et al. (2007) reported that the lactobacilli strain was generally susceptible to antibiotics that inhibit the synthesis of proteins, such as chloramphenicol, erythromycin, clindamycin, and tetracycline, and more resistant to aminoglycosides (neomycin, kanamycin, streptomycin, and gentamicin

| Hemolytic activity
Hemolysis was the rupture and dissolution of red blood cells, which can be caused by a variety of physical and chemical factors and toxins. Certain hemolytic Streptococcus and Bacillus perfringens can cause septicemia (Nami et al., 2019). The hemolytic test showed that Listeria monocytogenes as positive control showed a clear hemolytic ring and had β-hemolytic (Figure 3). The hemolytic activity test of L. plantarum DPUL-J5 and P. kudriavzevii DPUY-J5 showed negative results, indicating these strains were safe with no hemolytic activity.

| Physicochemical analyses of the fermented broad bean paste
The physicochemical parameters, including pH values, the content of total acids, reducing sugar, and amino acid nitrogen in four kinds of broad bean pastes fermented with different strains, were determined ( Figure 4). For the clarity of results, broad bean paste fermented by different strains was coded as sample AB, sample ABL, sample ABP, and sample ABLP, respectively.
The change in pH, total acid, reducing sugar, and amino nitrogen in broad bean paste were significant with the process of fermentation (p < .05). As shown in Figure 4a, the pH values of four kinds of broad bean paste samples fermented by different strains decreased, which was in accordance with the results of total acids concentration.
The pH value of sample ABL (4.83) was the lowest while sample ABP (4.98) had the highest pH value. LAB produced lactic acid, which was the major metabolic end product of carbohydrates in food fermentation processes that inhibited the growth of competing microbes (Smid & Kleerebezem, 2014). Fermented foods with a pH value below 4-5 were usually considered safe, as most of the pathogens were unable to survive at this pH value (Padonou et al., 2010   Amino acid nitrogen was usually considered as one of the main indices to represent broad bean paste quality. The protein in the broad bean was decomposed by microorganisms, resulting in the continuous increase in amino acid nitrogen. High amino acid nitrogen concentration might improve the quality of broad bean paste products. The amino acid nitrogen concentration was all increased since the start of fermentation and stabilized around 0.76 g/100 g, 0.67 g/100 g, 0.76 g/100 g, and 0.83 g/100 g for sample AB, ABL, ABP, and ABLP, respectively ( Figure 4d). The results showed that the four samples had the same upward trend during the fermentation period. Consistent with the results of this study, Choi and Bajpai, (2010) also found that the total amino acid nitrogen was low at the beginning of fermentation, and tended to increase slightly as fermentation proceeded.

| BA and AFB1 contents of broad bean paste
As shown in Figure (Shukla et al., 2014).
The results of AFB1 levels in broad bean paste samples were determined as shown in Figure 6. which exhibited ability to degrade aflatoxin B1 by 67.2%. Therefore, the starter culture was helpful to control the safety of broad bean paste fermentation when it did not produce AFB1 or degraded.

| Volatile flavor compounds in broad bean paste
A total of 42 volatile flavor compounds were detected in broad bean paste samples after 30 days of fermentation, including eight alcohols, six aldehydes, four ketones, thirteen esters, five phenols, two alkenes, one alkane, one pyrazine, and two acids. In sample AB, the main volatile flavor compounds were alcohols, esters, and aldehydes, while few other flavor substances were detected (Table 7). However, in the sample ABL and ABP, more ketones, phenols, and other types of flavor compounds were detected. We found that higher concentrations of alcohols were detected in the sample ABP because alcohols were mainly produced by yeast fermentation. The phenylethyl alcohol concentration reached 2309.90 ± 43.98 ng/g (p < .05); it was reported that phenylethyl alcohol give the sauce a strong floral aroma (Jelen et al., 2013). The types of volatile flavor compounds in sample ABL and ABP were significantly lower than those in sample ABLP, and the increase in ester flavor compounds was particularly obvious in group ABLP, which was caused by the cofermentation of yeast and lactic acid bacteria (Xie et al., 2018). In the ABLP group, phenols contributed greatly to the overall flavor of broad bean paste, the concentration of 2-methoxy-4-(1-propenyl)-phenol was noted to be 69.79 ± 8.75 ng/g (p < .05). Yin et al. (2022) found that 4-ethyl-guaiacol and 4-ethyl-phenol were the main phenolic compounds in chili paste, which were produced by the fermentation of Aspergillus and Pichia bulbar. The top three relative abundance of volatile flavor compounds in the fermentation broad bean paste were hexadecanoic acid-ethyl ester, linoleic acid-ethyl ester, and ethyl oleate. Hexadecanoic acid-ethyl ester and ethyl oleate had a wax-like odor and a floral scent, and linoleic acid-ethyl ester had a slight astringency in sausage fermentation (Zhao et al., 2011).
Thus, these compounds substantially contributed to the aroma of broad bean paste. Microorganisms played an important role in the fermentation of bean paste, LAB could provide the lactic acid, which together with yeast converted lactic acid and alcohols into esters, and aldehydes can be formed by the oxidation of alcohols and phenols (Yao et al., 2015). A large number of alcohols produced by yeast, or more aldehydes and ketones were reduced to alcohols during the fermentation (Zhao et al., 2021). Jeong et al. (2013) reported that the tastes, flavors, and qualities of kimchi were principally related to kimchi metabolites including organic acids, amino acids, mannitol, and carbohydrates, and the production of kimchi was influenced by the microbial community including LAB and yeasts present during fermentation. It is proved that the interaction of yeast and LAB can improve the types of volatile flavor compounds, but the quality of broad bean paste cannot be only judged by the types of volatile flavor compounds, and further quantitative identification of finished fermented products was needed. Our results showed that the aroma compounds in the broad bean paste were influenced by the presence of Lactobacillus plantarum DPUL-J5 and Pichia kudriavzevii DPUY-J5 during broad bean paste fermentation. Alcohols, esters, and aldehydes were the major flavor components in the broad bean paste.

| CON CLUS ION
Strains of Lactobacillus plantarum DPUL-J5 and Pichia kudriavzevii DPUY-J5 were screened from traditional Sichuan broad bean paste which produces almost no biogenic amines, has high protease activity, and shows no resistance to antibiotics and hemolytic activity.
The broad bean paste fermented by Lactobacillus plantarum DPUL-J5 and Pichia kudriavzevii DPUY-J5 was beneficial to the accumulation of total acid, amino acid nitrogen, and volatile flavor compounds while degrading harmful compound, such as biogenic amines. In a word, broad bean paste fermented with safe strains not only ensured the safety of the product but also improved the quality. Thus, this study provided reasonable suggestions for the standardization and safety of starter cultures in traditional broad bean paste production in Southwestern China.

ACK N OWLED G M ENT
This study was funded by the National Key Research and Development Plan (2018YFC1604102).

CO N FLI C T O F I NTE R E S T
The authors state no potential conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data are all included in the manuscript.